HPLC Column and System Troubleshooting 色谱柱和色谱系统的故

Pump 泵,Autosampler 自动进样器,Column 色谱柱,Detector 检测器,Integrator 积分仪

HPLC Column and System Troubleshooting

色谱柱和色谱系统的故障检修

What Every HPLC User Should Know

每个HPLC使用者应该知道什么2014-4-7 1

Pump 泵,Autosampler 自动进样器,Column 色谱柱,Detector 检测器,Integrator 积分仪

HPLC Components HPLC 的组成

Pump 泵 Injector/Autosampler 进样器/自动进样器 Column 色谱柱 Detector 检测器 Data System/Integrator 数据系统/积分仪表 All of these components can have problems and require troubleshooting. 所有这些部件可能出现故障而需要检修。2

2014-4-7

Pump 泵,Autosampler 自动进样器,Column 色谱柱,Detector 检测器,Integrator 积分仪

Categories of Column Problems 色谱 柱问题的类型

A. Pressure 压力 B. Peak shape 峰形 C. Retention 保留时间

2014-4-7

Pump 泵,Autosampler 自动进样器,Column 色谱柱,Detector 检测器,Integrator 积分仪

Pressure Issues 压力问题

Column Observations 色谱柱观测

Potential Problems 潜在的问题

High pressure 压力高

Plugged frit 堵塞滤头 Column contamination 色谱柱污染 Piugged packing 堵塞填料

2014-4-7

Pump 泵,Autosampler 自动进样器,Column 色谱柱,Detector 检测器,Integrator 积分仪

Determining the Cause and Correcting High Back Pressure 查出原因 纠正压力Check pressure with/without column - many pressure problems are due to blockages in the system or guard 有/无柱子时检查压力 - 许多压力问题是由于系统或保护柱的阻塞

If Column Pressure is high 如果柱压高:Wash column - Eliminate column contamination and

冲洗柱子

plugged packing 排除柱污染和填料堵塞 - high molecular weight/adsorbed compounds 高分子量/被吸附化合物 - precipitate from sample or buffer 样品或缓冲液中的沉淀物

Back flush column - Clear plugged frit 反冲柱子-清洁阻塞的滤头 Change frit - Clear plugged frit 更换滤头5

2014-4-7

Pump 泵,Autosampler 自动进样器,Column 色谱柱,Detector 检测器,Integrator 积分仪

Column Cleaning 柱子清洗

F lush with stronger solvents than your mobile phase

用比你的流动相更强的溶剂冲洗Reversed-Phase Solvent Choices in Order of Increasing Strength 为了增加强度选择反相溶剂Use at least 25 ml of each solvent for analytical columns 对于分析柱每种溶剂至少用25 ml 冲洗Mobile phase without buffer salts 没有缓冲盐的流动相 100% Methanol 甲醇 100% Acetonitrile 乙腈 75% Acetonitrile :25% Isopropanol 75乙腈 ： 25%异丙醇 100% Isopropanol 异丙醇 100% Methylene Chloride★ 二氯甲烷 100% Hexane ★ 已烷★

When using either Hexane or Methylene Chloride the column must be flushed with

Isopropanol before returning to your resvered-phase mobile phase.

当用已烷蔌二氯甲烷柱子时,必须先用异丙醇冲洗,然后再用你的反相流动相2014-4-7 6

Pump 泵,Autosampler 自动进样器,Column 色谱柱,Detector 检测器,Integrator 积分仪

Column Cleaning柱子清洗

Normal Phase Solvent Choices in Order of Increasing Strength为了增加强度选用正相溶剂Use at least 50 ml of each solvent 每种溶剂至少用50 ml50% Methanol : 50% Chloroform 50% 甲醇 : 50% 氯仿 100% Ethyl Acetate 乙酸乙酯7

2014-4-7

Pump 泵,Autosampler 自动进样器,Column 色谱柱,Detector 检测器,Integrator 积分仪

How to Change a Frit 怎样更换滤头

Column Inlet Frit 柱进口滤头 Column Body 柱身 Compression Ferrule 压缩金属套圈Wear gloves 戴手套 Do not allow bed to dry 不要让底座干燥 Do not touch the column-body heat will extrude packing 不要使柱身受热,否则填料会喷出 Do not over tighten 不要旋得太紧

Female End Fitting 旋好尾端螺母 Male End Fitting 旋好尾端螺头2014-4-7 8

Pump 泵,Autosampler 自动进样器,Column 色谱柱,Detector 检测器,Integrator 积分仪

Preventing Back Pressure Problems

压力问题的预防

Use column protection 柱保护- Guard columns 保护柱/预柱

- In-line filters 在线过滤器

Sample Preparation 样品预处理 Appropriate column flushing 适当的柱冲洗 Filter buffered mobile phase 过滤缓冲流动相9

2014-4-7

Pump 泵,Autosampler 自动进样器,Column 色谱柱,Detector 检测器,Integrator 积分仪

Preventing Back Pressure Problems:In-Line Devices

压力问题的预防:在线装置Pre-Column 预柱

Mobile Phase From Pump 从泵来的流动相

Injector Filter Guard 进样器 滤头 Column 保护柱

Analytical Column Filter and Guard Column Act on Sample 分析柱 滤头和保护柱作用于样品 Pre-Column Acts on Mobile Phase 预柱作用于流动相 To Detector 到检测器

2014-4-7

Pump 泵,Autosampler 自动进样器,Column 色谱柱,Detector 检测器,Integrator 积分仪

Preventing Back Pressure Problems: Sample Preparation

压力问题的预防:样品制备

Solvent/Chemical Environment 溶剂/化学环境 Particulate/Aggregate Remove 微粒/凝聚物的除去Filter samples 过滤样品 Centrifugation 离心分离

Solid Phase Extraction(S.P.E.) 固相萃取Cartridges or Plates 薄膜或薄层板 Disks or Membranes 圆盘或生物膜11

2014-4-7

Pump 泵,Autosampler 自动进样器,Column 色谱柱,Detector 检测器,Integrator 积分仪

Peak Shape Issues 峰形问题Split peaks 裂峰 Peak tailing 峰拖尾 Broad peaks 宽峰 Poor efficiency 低柱效

Many peak shape issues also combinations-I.e. Broad and tailing or tailing with increased retention许多峰形问题 是结合在一起的-例如: 展宽和拖尾或拖尾和保留时间增加12

2014-4-7

Pump 泵,Autosampler 自动进样器,Column 色谱柱,Detector 检测器,Integrator 积分仪

Split Peaks 裂峰 Can

be caused by: 可能的原因:

Column contamination 柱污染 Partially plugged frit 部分阻塞滤片 Column void 柱头塌陷 Injection solvent effects 溶剂效应13

2014-4-7

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